In Focus pubs.acs.org/acschemicalbiology
Chemical Biology of Nucleic Acids Jan Barciszewski,† Victor E. Marquez,‡ Jean-Jacques Vasseur,§ and Wojciech T. Markiewicz*,† †
Institute of Bioorganic Chemistry of the Polish Academy of Sciences, 61-704 Poznań, Poland Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, Maryland 21702, United States § Institut des Biomolécules Max Mousseron, University Montpellier, 33095 Montpellier, France ‡
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all classes of noncoding RNAs) and the extent to which various steps in nucleic acids biosynthesis and processing are mechanistically intertwined. The general feature of any DNA and RNA technologies is the programmability of nucleic acid structure using always the same Watson−Crick base pairs that are responsible for the information content of all living systems. Therefore, the secondary structure recognition interactions are reliable, reversible, and responsive, with a low mistakes rate. The existence and success of the field is clearly a consequence of the convenient availability of specific sequences of nucleic acids, owing to the development of phosphoramidite chemistry in the early 1980s. As can be seen in many of the articles, DNA structures, although robust, are not necessarily static constructs. In addition to the well-known Watson−Crick double helix, nucleic acids adopt a number of unusual motifs, at least in vitro. The biological relevance of these noncanonical structures has often been questioned as nonphysiological conditions are often used to obtain them. RNA can in contrast to DNA accelerate chemical reactions by 1 million fold or more. RNA has become a focus of investigations into novel therapeutic schemes. Ribozymes, antisense RNAs, RNA decoys, aptamers, microRNA, and small interfering RNAs (siRNAs) have been used to downregulate undesired gene expression (Figure 1). It turned out that small RNAs are short noncoding regulatory RNAs that direct gene silencing in a sequence-dependent manner. More than 20 years have passed since the first
he XXI International Round Table (IRT) on Chemical Biology of Nucleic Acids was held in Poznań, Poland, August 24−28, 2014. It attracted 400 scientists from 32 countries. Various aspects of chemistry and biology of nucleic acids and their components were discussed. There were 23 invited talks and 33 oral presentations covering topics ranging from chemical synthesis of nucleic acids to their application as drugs and tools. The meeting highlighted recent advances in DNA and RNA chemistry, biology, biochemistry, and biophysics and identified emerging concepts of their application in molecular medicine. Many talks presented at the meeting illustrated ways to approach biological properties to answer both general and detailed questions in chemical biology of nucleosides, nucleotides, and nucleic acids. More than 250 posters were presented. The poster session offered an opportunity for undergraduate Ph.D. students, postdoctoral fellows, and early career researchers to present their results and interact together and with senior scientists. To acknowledge outstanding scientific results of these young scientists, several “IS3NA, A. Holý IRT Poster Awards,” sponsored by Gilead Sciences were distributed in honor of Professor Antonı ́n Holý (1936−2012) as a tribute to his outstanding career and involvement in the development of antiretroviral drugs. Among 110 eligible posters (for candidates having less than five years after their Ph.D.), 12 posters were selected for the awards. The International Round Table on Nucleic Acids series was first established more than 40 years ago. The first IRT on Nucleosides, Nucleotides, and Nucleic Acids was held in Montpellier in 1974. Since then, the field of nucleic acids has exploded with tremendous discoveries regarding the chemistry, functional potential, and biologic significance. Nucleic Acids IRT conferences provide a critical forum for discussion of new ideas by promoting interactions between investigators specializing in all diverse areas of nucleic acids research. It is obvious that nucleic acids are fundamental to the understanding of life. As soon as J.D. Watson and F.H.C Crick proposed the DNA structure 61 years ago, thoughts of many chemists and biologists switched not only to DNA but also RNA. The other milestone marking its 50th anniversary this year was H.G. Khorana’s and co-workers’ achievements in the chemical synthesis of RNA triplets as the foundation to genetic code “cracking.” Nowadays in the era of whole genomes, chemical biology prompts scientists to understanding biochemical and biological properties of nucleic acids. The past decade has brought renewed recognition and excitement regarding the breadth of nucleic acid functions in cells (i.e., © 2015 American Chemical Society
Figure 1. Possible ways for protein synthesis regulation. There are two groups of approaches: gene silencing and protein inhibition. Published: June 19, 2015 1358
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In Focus
ACS Chemical Biology
Figure 2. Types of modified nucleic acids.
Figure 3. Basic and modified DNA and RNA: 2′-OMe-RNA, LNA, UNA, and PNA.
microRNA (miRNA) was discovered. Since then, small RNAmediated gene silencing has set a new paradigm for understanding eukaryotic gene regulation (Figure 1). Multiple challenges, such as optimization of selectivity, stability, delivery, and long-term safety, have to be addressed in order for RNA drugs to become a successful therapeutic category (Figure 2). Generally, there are five known types of modified nucleic acids. All of them show higher stability within the cell. A new type of catalytic nucleic acids was presented at the IRT. They are based on L-ribose (RNA) or L-deoxyribose (DNA). New catalytic nucleic acids (L-hammerhead-zymes and
L-DNA-zymes)
have been synthesized (V.A. Erdmann, Berlin). They show an identical spectrum of enzymatic activity to DDNA-zymes, but they are much more stable. For the first time, mirror image hammerhead ribozyme or DNA-zyme were used in living cells for the targeted cutting of natural nucleic acids.1 These molecular scissors are stable, and they do not appear to trigger side reactions of the immune system. It has been proposed that heterochiral double helix structures can be formed by Watson−Crick base pairs where nucleotides occur in an anticline conformation. Furthermore, the heterochiral 1359
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In Focus
ACS Chemical Biology duplexes form either right- of left-handedness for D- or Lribozyme, respectively.2 Many facets of RNA are more and more understood, and now it is possible to design and use various RNAs as valuable tools in molecular biology and medicine for diagnosis, design, and development of drugs and new therapeutics for fighting cancers, viruses, and other diseases at the nucleic acids level. The stability of these constructs is ensured with incorporation of different modified nucleic acids units (Figure 3). One of the topics broadly discussed was quadruplexes. They are a family of nucleic acid structures based on the formation of G-quartets, a planar arrangement of four guanines that establish a cyclic arrangement of hydrogen bonds. A central spine of cations, such as potassium or sodium, stabilizes the resulting stacking between consecutive G-quartets. The self-assembly properties of guanosine derivatives have been known for over a century, and the tetrahelical structure of GMP was established more than 50 years ago. Nucleosides are very resilient, and they continue to form an important component of the armamentarium of drugs approved by the FDA. For most nucleosides, the validated target is the viral polymerase, and since pol sites are very similar, nucleosides can attack a myriad of viruses. The successful past of nucleoside drugs, beginning with the game changer drug “Acyclovir” to the present blockbuster hepatitis C drug Sofosbuvir for the treatment of most HCV genotypes, was discussed (D. Liotta, Emory University). Sofosbuvir and other nucleosides under development are given as prodrugs capable of bypassing the first cellular phosphorylation step and delivering the nucleoside 5′-monophosphate. For these drugs to act, as in the case of Sofosbuvir, some nonubiquitous phosphoramidases are required, thus presenting a challenge for drug development. Another important aspect to consider is the half-life of the active 5′-triphosphate, which in the case of the anti-HIV L-cytosine nucleoside analog, Elvucitabine, is quite long and leads to toxicity. In these cases, reduction of the frequency of dosing solves the problem.3 From more than 350 nucleoside analogues and 40 prodrug scaffolds that produced 2800 prodrugs (diastereoisomers that were separated), 220 were investigated for the formation of the 5′-triphosphates in mice. A new drug was discovered (IDX 21437) that showed potent antiviral activity against HCV genotypes 1, 2, or 3 in patients receiving 300 mg once daily. That resulted in a 4.2 log reduction in viral load after 7 days of treatment. The drug is selective for hepatocytes, and levels of 5′-triphosphate in monkey and mouse livers are 5 times higher than those achieved with Sofosbuvir. Furthermore, the drug is not metabolized by cardiomyocytes (C.B. Dousson, Montpellier).4 Clinically, Favipiravir (T-705), and its T-705 ribofuranosyltriphosphate (T-705 RTP) inhibits the influenza A virus RNA polymerase by acting as a mimic of ATP and GTP. Two consecutive incorporations of T-705 RTP lead to chain termination. On the other hand, 2′-fluoro-GTP is a leaky chain terminator for IAV polymerase and incorporated by human mitochondrial RNA and DNA polymerase, and it cannot be advanced clinically. With aim to improve potency and reduce toxicity, syntheses of novel 2′-fluoro-4′-thio-purine nucleosides were pursued in this investigation; while the final compound was not a substrate to human polymerase, it also lost activity against target viral polymerase (V.K. Rajwanshi, L. Beigelman, Alios BioPharma).5
The cycloSal-prodrug approach has already been successfully applied to the development of nucleoside monophosphate prodrugs. The more challenging delivery of nucleoside di- and triphosphates by di- or triphosphate prodrugs was presented. The surprising concept here is that not all the negative charges on the di- or triphosphate system need to be masked. Prodrugs of a well-known anti-HIV nucleoside analogue (d4T), which was used for the design of effective di- and triphosphate prodrugs, were presented. The success of the design was based on tuning lipophilicity versus charge. This was accomplished by only masking the beta or gamma phosphate and leaving the alpha or the alpha and beta phosphates unmasked. For the synthesis of the triphosphate prodrugs the tri-n-butyl ammonium salt of d4TDP, prepared via the cycloSal-approach, was reacted with bis(4-(acyloxybenzyl)phosphoramidites to give after oxidation the target prodrug which upon incubation with pig liver esterase or CEM cell extracts delivered the desired triphosphate. In the case of d4T, the antiviral activity of the triphosphate prodrugs in wild-type CEM cells as well as in thymidine kinases-deficient cells was very good but was dependent on the length of the lipophilic acyl chain (C. Meier, Hamburg).6 DNA methylation and histone methylation are both involved in epigenetic regulation of gene expression and their dysregulation can play an important role in leukemogenesis. Aberrant DNA methylation has been reported to silence the expression of tumor suppressor genes in leukemia, and overexpression of the histone methyltransferase (EZH2), a subunit of the polycomb group repressive complex 2 (PRC2), is known to promote oncogenesis. Epigenetic changes are more frequent than genetic mutations leading to cancer and have the advantage of being reversible and even subject to drug treatment. Exploiting the cross talk that exists between these epigenetic events was achieved by using combinations of a DNA methylation inhibitor (5-aza-2′-deoxycytidine, 5-AdC) and a histone methylation inhibitor (3-deazaneplanocin A, DZNep). Microarray analysis showed that 5-AdC plus DZNep produced a synergistic upregulation of >1000 genes related to apoptosis which increased from 3% to 45% above control values. Resting cells were not affected. Best results are obtained when the drugs are used sequentially starting with 5-AdC followed by DZNep after 24 h (R.L. Momparler, Montreal).7 4′-Selenonucleosides appear to open a new frontier for the development of novel cancer treatments. The heavy Se atom, for example, imparts the South conformation to 4′selenocytidine and 4′-selenouridine, which contrasts with the North conformation of cytidine and uridine.8 Rearranging the stereochemistry of the sugar OH groups resulted in the corresponding 4′-seleno-ara-C, which showed excellent anticancer activity.9 An ensuing structure−activity study revealed that changing the OH for a F, which was introduced with the ara stereochemistry by a double inversion with the fluorinating reagent DAST, produced an even more potent compound,10 whose mechanism of action does not appear to be via the formation of the corresponding 5′-triphosphate, but rather by targeting the c-Met tyrosine kinase.11 C-Met inihibitors are a class of compounds that have therapeutic applications in the treatment of cancer (L.S. Jeong, Seoul). Gemcitabine is a well-established anticancer agent used in the clinic. Because clinical studies have indicated that prolonged infusion times with lower doses of Gemcitabine can be effective while reducing toxicity to normal cells, several prodrugs have been developed featuring N-alkyl/acyl modifications. The 1360
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ACS Chemical Biology syntheses of various Gemcitabine analogues with 4-N-alkanoyl and 4-N-alkyl chains modified with a terminal hydroxyl, halide, or alkene groups were reported. The 4-N-alkyl analogues are stable toward deamination and were studied for their anticancer activity as well as for their possible use in Positron Emission Tomography (PET) scans. All 4-N-alkanoyl analogues demonstrated potent antiproliferative activities with IC50 values in the low nanomolar range, similar to Gemcitabine. However, 4-N-alkylgemcitabine derivatives were only modestly active. Some of the N-alkanoyl analogues with a terminal 18F labeling were prepared expediently and are expected to be suitable for PET scanning (S.F. Wnuk, Miami).12 Pseudouridine is a constituent nucleoside of tRNA, and increased levels of this nucleoside in urine could represent novel biomarkers for colorectal cancer diagnosis and surgery monitoring. In order to develop a portable device based a bulk acoustic wave molecularly imprinted polymer sensor (SAWMIP), a noncovalent imprinting polymer with a robust and stable binding site for pseudouridine was reported. The 2,6-bisacrylamidopyridine monomer is bound to pseudouridine through H-bonding, and polymerization and cross-linking by microwave heating resulted in the formation of the active site. Retention levels of 96% for pseudouridine were achieved (A. Krstulja, L.A. Agrofoglio, Orleans).13 As opposed to Spherical Nucleic Acids (SNA) constructed by using a 3′-alkanethiol-terminated single-stranded DNA covalently attached to a gold nanoparticle, these SNAs are made with lipid-tagged oligonucleotides embedded into a lipid bilayer of a micelle or liposome-type structure. These lipo SNAs are recognized by Toll-Like Receptors (TRL) which are a class of proteins that play a key role in the immune system. These lipo SNAs increase interleukin 12 (IL-12) levels ca. 20fold more than the corresponding free oligonucleotides. They are capable of bringing a special 3D orientation of the pharmacophore in space that increased by 1000-fold the production of antibodies (S.M. Gryaznov, AuraSense).14 Nucleoside 1,2,3-Triazoles as antiviral agents are synthesized by clicking (Huisgen cycloaddition) AZT to bulky alkynes. For HIV, the corresponding triphosphates showed lower ATPmediated nucleotide excision efficiency compared to AZT, which along with molecular modeling suggests a mechanism of preferred translocation of triazoles into the P-site of HIV reverse transcriptase (RT). This event overcomes resistance to an AZT-resistant variant.15 In addition, similar nucleoside 1,2,3triazole compounds also inhibited West Nile virus (WNV) and Dengue virus (DENV), likely via targeting viral methyl transferase.16 Remarkably there is no overlap in anti-HIV SAR and antiflaviviruses (WNV and DENV) SAR as activity against HIV-1 entails a free 5′-hydroxyl group while potency against WNV or DENV requires silyl protection of the 5′hydroxyl group (Z. Wang, Minnesota).15,16 The IMP preferring cytosolic 5′-nucleotidase (cN-II) is a ubiquitous nucleotide-hydrolyzing enzyme. It is highly expressed in several tumor cells, and overexpression is often associated with resistance phenomena. This feature is reinforced by the recent discovery of hyperactive mutants of cN-II inducing similar response. S. Peyrottes (Montpellier, France) and co-workers, proposed the development of nucleoside phosphonates as potential inhibitors of this enzyme. SAR studies showed that phosphonate analogues bearing (i) a (S)-hydroxy group at the C-5′ position, (ii) a ribo- or an araconfiguration of the sugar moiety, and (iii) a cytosine as nucleobase were required for activity.17,18
In conclusion, we would like to say that nucleic acid research follows Richard Feynman’s famous statement “What I cannot create, I do not understand,” which strongly stimulates a basic science and greater understanding of biology and chemistry of life.
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AUTHOR INFORMATION
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(1) Wyszko, E., Szymański, M., Zeichhardt, H., Mü ller, F., Barciszewski, J., and Erdmann, V. A. (2013) Spiegelzymes: Sequence Specific Hydrolysis of L-RNA with Mirror Image Hammerhead Ribozymes and DNAzymes. PLoS One 8, e54741. (2) Wyszko, E., Mueller, F., Gabryelska, M., Bondzio, A., Popenda, M., Barciszewski, J., and Erdmann, V. A. (2014) Spiegelzymes® Mirror-Image Hammerhead Ribozymes and Mirror-Image DNAzymes, an Alternative to siRNAs and microRNAs to Cleave mRNAs In Vivo? PLoS One 9, e86673. (3) Liotta, D. Unpublished work. (4) Dousson, C. B. Unpublished work. (5) Rajwanshi, V. K. Unpublished work. (6) Meier, C., Gollnest, T., Weinschenk, L., Balzarini, J. Unpublished work. (7) Momparler, R. L., Idaghdour, Y., Marquez, V. E., and Momparler, L. F. (2012) Synergistic antileukemic action of a combination of inhibitors of DNA methylation and histone methylation. Leuk. Res. 36, 1049−1054. (8) Jeong, L. S., Tosh, D. K., Hea, O. K., Wang, T., Hou, X., Ho, S. Y., Kwon, Y., Sang, K. L., Choi, J., and Long, X. Z. (2008) First synthesis of 4′-selenonucleosides showing unusual southern conformation. Org. Lett. 10, 209−212. (9) Jeong, L. S., Tosh, D. K., Won, J. C., Sang, K. L., Kang, Y. J., Choi, S., Jin, H. L., Lee, H., Hyuk, W. L., and Hea, O. K. (2009) Discovery of a new template for anticancer agents: 2′-deoxy-2′- fluoro-4′selenoarabinofuranosyl-cytosine (2′-F-4′-seleno-ara-C). J. Med. Chem. 52, 5303−5306. (10) Kim, J. H., Yu, J., Alexander, V., Choi, J. H., Song, J., Lee, H. W., Kim, H. O., Choi, J., Lee, S. K., and Jeong, L. S. (2014) Structureactivity relationships of 2′-modified-4′- selenoarabinofuranosyl-pyrimidines as anticancer agents. Eur. J. Med. Chem. 83, 208−225. (11) Jeong, L. S. Unpublished work. (12) Pulido, J., Sobczak, A. J., Balzarini, J., and Wnuk, S. F. (2014) Synthesis and cytostatic evaluation of 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues. J. Med. Chem. 57, 191−203. (13) Krstulja, A. and Agrofoglio, L. A. Unpublished work. (14) Gryaznov, S. M. Unpublished work. (15) Sirivolu, V. R., Vernekar, S. K. V., Ilina, T., Myshakina, N. S., Parniak, M. A., and Wang, Z. (2013) Clicking 3′-Azidothymidine into Novel Potent Inhibitors of Human Immunodeficiency Virus. J. Med. Chem. 56, 8765−8780. (16) Vernekar, S. K. V.; Qiu, L.; Kankanala, J.; Zhang, J.; Li, H.; Geraghty, R. J.; Wang, Z. 5′-Silylated nucleoside scaffolds as inhibitors of West Nile virus and Dengue virus. Manuscript submitted. (17) Gallier, F., Lallemand, P., Meurillon, M., Jordheim, L. P., Dumontet, C., Périgaud, C., Lionne, C., Peyrottes, S., and Chaloin, L. (2011) Structural insights into the inhibition of cytosolic 5′nucleotidase II (cN-II) by ribonucleoside 5′-monophosphate analogues. PLoS Comput. Biol. 7, e1002295. (18) Meurillon, M., Marton, Z., Hospital, A., Jordheim, L. P., Béjaud, J., Lionne, C., Dumontet, C., Périgaud, C., Chaloin, L., and Peyrottes, S. (2014) Structure-activity relationships of β-hydroxyphosphonate nucleoside analogues as cytosolic 5′-nucleotidase II potential inhibitors: Synthesis, in vitro evaluation and molecular modeling studies. Eur. J. Med. Chem. 77C, 18−37.
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